Pipette-tip kapok fiber-based solid-phase extraction/in-situ derivatization for the rapid and green analysis of furfural compounds.

Furfural compounds, including 5-hydroxymethylfurfural, furfural, and 5-methylfurfural, are common in foods and pose health risks. This study presents a pipette-tip solid-phase extraction with in-situ derivatization (PT-KF-SPE/ISD) method for rapid analysis of furfural compounds in various food matrices. Utilizing natural kapok fiber as an efficient adsorbent, this method integrates extraction and derivatization into a single step via a simple pull-push operation. Derivatization with 2,4-dinitrophenylhydrazine increases the hydrophobicity and ultraviolet absorption of furfural compounds, enabling sensitive liquid chromatography-ultraviolet detection. The method shows good linearity, sensitivity, and reproducibility, with limits of detection in ranges of 3.9-6.0 ng/mL. Real sample analysis confirms its applicability in detecting furfural compounds in beverages and herbal products, offering a reliable and eco-friendly solution for food safety and quality control. Five greenness assessment metrics demonstrate the method's excellent environmental friendliness. This approach highlights the advantages of combining natural adsorbents with in-situ derivatization for efficient food analysis.

Development of the CRISPR-Cas12a system for editing of Pseudomonas aeruginosa phages.

Pseudomonas aeruginosa is a common opportunistic pathogen. The potential efficacy of phage therapy has attracted the attention of researchers, but efficient gene-editing tools are lacking, limiting the study of their biological properties. Here, we designed a type V CRISPR-Cas12a system for the gene editing of P. aeruginosa phages. We first evaluated the active cutting function of the CRISPR-Cas12a system in vitro and discovered that it had a higher gene-cutting efficiency than the type II CRISPR-Cas9 system in three different P. aeruginosa phages. We also demonstrated the system's ability to precisely edit genes in Escherichia coli phages, Salmonella phages, and P. aeruginosa phages. Using the aforementioned strategies, non-essential P. aeruginosa phage genes can be efficiently deleted, resulting in a reduction of up to 5,215 bp (7.05%). Our study has provided a rapid, efficient, and time-saving tool that accelerates progress in phage engineering.

Characterization, genome analysis, and therapeutic evaluation of a novel Salmonella phage vB_SalS_JNS02: a candidate bacteriophage for phage therapy.

Phage therapy is gaining momentum as an alternative to antibiotics in the treatment of salmonellosis caused by Salmonella. In this study, a novel Salmonella phage, vB_SalS_JNS02, was isolated successfully from poultry farms in Shandong, China. The biological characteristics of vB_SalS_JNS02 were analysed, which revealed a short latent period of approximately 10 min and a burst size of 110 PFU/cell. Moreover, vB_SalS_JNS02 exhibited remarkable stability across a wide pH range (pH 3-12) and temperatures ranging from 30 to 80°C. Genome sequencing analysis provided valuable insights into the genetic composition of vB_SalS_JNS02, which consists of a double-stranded DNA genome that spans 42,450 base pairs and has a G + C content of 49.4%. Of significant importance, the genomic sequence of vB_SalS_JNS02 did not contain any genes related to lysogenicity, virulence, or antibiotic resistance. The phage's efficacy was evaluated in a larval challenge study. Treatment with the phage resulted in increased survival of Galleria mellonella larvae (100, 70, and 85%) (MOI 0.1) in the prophylactic treatment, co-infection treatment, and remedial treatment experiments, respectively. Another in vivo experiment investigated the potential application of the phage in broiler chickens and revealed that a single oral dose of vB_SalS_JNS02 (108 PFU/mL, 100 µL/chick) administered 3 h after S. enteritidis oral administration provided effective protection. The introduction of bacteriophage not only enhances the production of secretory immunoglobulin A (sIgA), but also induces alterations in the composition of the gut microbial community. Phage therapy increases the relative abundance of beneficial bacteria, which helps to maintain intestinal barrier homeostasis. However, it is unable to fully restore the disrupted intestinal microbiome caused by S. enteritidis infection. Importantly, no significant adverse effects were observed in the animal subjects following oral administration of the phage, and our findings highlight vB_SalS_JNS02 is a hopeful candidate as a promising tool to target Salmonella infections in poultry.

Antimicrobial Resistance and Molecular Characterization of Escherichia coli from Healthy Chickens in Shandong, China from 2009 to 2014.

Antimicrobial resistance (AMR) is a great threat to animal and public health. Here, we conducted a surveillance of Escherichia coli isolated from healthy chickens during 2009-2014 to identify the characteristics of AMR. A total of 351 (95.64%) E. coli isolates were obtained from 367 healthy chicken fecal samples collected from 6 farms located in Shandong Province, China. The susceptibility to 10 antimicrobials, the prevalence of antibiotic resistance genes (ARGs), phylogenetic clustering, and multilocus sequence typing were evaluated. The isolates exhibited high resistant rates (>95%) to ampicillin, cefotaxime, ciprofloxacin, ceftiofur, and enrofloxacin. The most prevalent ARGs were blaCTX-M (36.36%), aac(6')-Ib-cr (30.79%), qnrS (29.62%), oqxAB (27%), mcr-1 (15.83%), blaTEM (9.09%), qnrC (3.52%), qnrD (0.88%), and qepA (0.29%). Phylogenetic clustering analysis indicated that the most prevalent group was group D (37.89%), followed by group B1 (34.76%), A (24.22%), and B2 (3.13%). Fifty-seven sequence types (STs) were identified among the 124 blaCTX-M-positive strains, and the dominant STs were ST354 (13.71%), ST117 (5.65%), ST155, ST2309, and ST2505 (4.84% each). There was a significant association between 17 pairs of AMR phenotypes, 14 pairs of ARGs, and 11 pairs of AMR-ARGs. The strongest association was found between ST602 and qnrC (odds ratios: 22.2). This study implied that E. coli isolated from healthy chickens could potentially serve as a reservoir of AMR and ARGs, and significant associations exist among AMR, ARGs, phylogenetic groups, and STs. Our study highlighted the need for routine surveillance of AMR in healthy chickens, and promoting appropriate antibiotic use and implementing regular monitoring of resistance in broilers are crucial for fostering the development of the poultry industry and safeguarding public health.

Toxin gene detection and antibiotic resistance of Clostridium perfringens from aquatic sources.

Clostridium perfringens is a zoonotic opportunistic pathogen that produces toxins that can cause necrotic enteritis and even "sudden death disease". This bacterium is widely distributed in the intestines of livestock and human, but there are few reports of distribution in aquatic animals (Hafeez et al., 2022). In order to explore the isolation rate of C. perfringens and the toxin genes they carry, 141 aquatic samples, including clams (Ruditapes philippinarum), oysters (Ostreidae), and mud snails (Bullacta exerata Philippi), were collected from the coastal areas of Shandong Province, China. C. perfringens strains were tested for cpa, cpb, etx, iap, cpb2, cpe, netB, and tpeL genes. 45 clam samples were boiled at 100 °C for 5 min before bacteria isolation. 80 strains were isolated from 141 samples with the positive rate being 57 %.And the positive rates of cooked clams was 87 % which was higher than the average. In detection of 8 toxin genes, all strains tested cpa positive, 3 strains netB positive, and 2 cpb and cpe, respectively. 64 strains were selected to analyze the antibiotic resistance phenotype of 10 antibiotics. The average antibiotic resistance rates of the strains to tetracycline, clindamycin, and ampicillin were 45 %, 20 %, and 16 % respectively, and the MIC of 4 strains to clindamycin was ≥128 µg/mL. A high isolation rate of C. perfringens from aquatic animals was shown, and it was isolated from boiled clams for the first time, in which cpe and netB toxin genes were detected for the first time too. The toxin encoded by cpe gene can cause food poisoning of human, thus the discoveries of this study have certain guiding significance for food safety. Antibiotics resistant C. perfringens of aquatic origin may arise from transmission in the terrestrial environment or from antibiotic contamination of the aquaculture environment and is of public health significance.

Genomic analysis and experimental pathogenic characterization of Riemerella anatipestifer isolates from chickens in China.

Waterfowl have a high likelihood of being infected with Riemerella anatipestifer. Although the pathogen is found in domestic ducks, turkeys, geese, and wild birds, there is little information available about the consequences of infection during egg laying and hatching in chickens. Here, we present the first report of a novel sequence type of R. anatipestifer S63 isolated from chickens in China. On the basis of pan-genome analysis, we showed S63's genome occupies a distinct branch with other R. anatipestifer isolates from other hosts. Galleria mellonella larval tests indicated that S63 is less virulent than R. anatipestifer Ra36 isolated from ducks. Ducks and hens are susceptible to S63 infection. There is no mortality rate for chickens or ducks, but adult chickens experience neurological symptoms that reduce egg production and hatching rates. In chickens, S63 might be passed vertically from parents to offspring, resulting in "jelly-like" lifeless embryos. Using quantitative PCR, S63 was detected in the brain, liver, reproductive organs, and embryos. As far as we know, this is the first report of R. anatipestifer in hens, a disease that can reduce egg productivity, lower hatching rates, and produce jelly-like lifeless embryos, and the first report to raise the possibility that hens can be infected by roosters via semen.

Villin headpiece unfolding upon binding to boridene mediated by the "anchoring-perturbation" mechanism.

We employ molecular dynamics (MD) simulations to investigate the influence of boridene on the behavior of a protein model, HP35, with the aim of assessing the potential biotoxicity of boridene. Our MD results reveal that HP35 can undergo unfolding via an "anchoring-perturbation" mechanism upon adsorption onto the boridene surface. Specifically, the third helix of HP35 becomes tightly anchored to the boridene surface through strong electrostatic interactions between the abundant molybdenum atoms on the boridene surface and the oxygen atoms on the HP35 backbone. Meanwhile, the first helix, experiencing continuous perturbation from the surrounding water solution over an extended period, suffers from potential breakage of hydrogen bonds, ultimately resulting in its unfolding. Our findings not only propose, for the first time to our knowledge, the "anchoring-perturbation" mechanism as a guiding principle for protein unfolding but also reveal the potential toxicity of boridene on protein structures.

Phage Tail Fiber Protein as a Specific Probe for Recognition of Shiga Toxin-Producing Escherichia coli O91, O103, and O111.

The ability to quickly identify specific serotypes of Shiga toxin-producing Escherichia coli (STEC) could facilitate the monitoring and control of STEC pathogens. In this study, we identified the receptors and receptor-binding proteins (RBPs) of three novel phages (pO91, pO103, and pO111) isolated from hospital wastewater. Recombinant versions of these RBPs (pO91-ORF43, pO103-ORF42, and pO111-ORF8) fused to a fluorescent reporter protein were then constructed. Both fluorescence microscopy and transmission electron microscopy showed that all three recombinant RBPs were bound to the bacterial surface. Indirect enzyme-linked immunosorbent assay was used to verify that each recombinant RBP bound specifically to E. coli O91, O103, or O111, but not to any of the 83 strains of E. coli with different O-antigens, nor to 10 other bacterial species that were tested. The recombinant RBPs adsorbed to their respective host bacteria within 10 min of incubation. The minimum concentration of bacteria required for detection by the recombinant RBPs was 33 colony-forming units (CFU)/mL (range: 3.3 × 10 to 3.3 × 108 CFU/mL). Furthermore, each recombinant RBP was also able to detect bacteria in lettuce, chicken breast meat, and infected mice, indicating that their usage will facilitate the detection of STEC and may help to reduce the spread of STEC-related infections and diseases.

Application of the Intelligent High-Throughput Antimicrobial Sensitivity Testing/Phage Screening System and Lar Index of Antimicrobial Resistance.

To improve the efficiency of antimicrobial susceptibility testing (AST) and phage high-throughput screening for resistant bacteria and to reduce the detection cost, an intelligent high-throughput AST/phage screening system, including a 96-dot matrix inoculator, image acquisition converter, and corresponding software, was developed according to AST criteria and the breakpoints of resistance (R) formulated by the Clinical & Laboratory Standards Institute (CLSI). AST and statistics of minimum inhibitory concentration (MIC) distributions (from R/8 to 8R) of 1,500 Salmonella strains isolated from poultry in Shandong, China, against 10 antimicrobial agents were carried out by the intelligent high-throughput AST/phage screening system. The Lar index, meaning "less antibiosis, less resistance and residual until little antibiosis", was obtained by calculating the weighted average of each MIC and dividing by R. This approach improves accuracy in comparison with using the prevalence of resistance to characterize the antimicrobial resistance (AMR) degree of highly resistant strains. For the strains of Salmonella with high AMR, lytic phages were efficiently screened from the phage library by this system, and the lysis spectrum was computed and analyzed. The results showed that the intelligent high-throughput AST/phage screening system was operable, accurate, highly efficient, inexpensive, and easy to maintain. Combined with the Shandong veterinary antimicrobial resistance monitoring system, the system was suitable for scientific research and clinical detection related to AMR.

Moderate binding of villin headpiece protein to C3N3 nanosheet reveals the suitable biocompatibility of this nanomaterial.

Since its recent successful synthesis and due to its promising physical and chemical properties, the carbon nitrite nanomaterial, C3N3, has attracted considerable attention in various scientific areas. However, thus far, little effort has been devoted to investigating the structural influence of the direct interaction of this 2D nanomaterial and biomolecules, including proteins and biomembranes so as to understand the physical origin of its bio-effect, particularly from the molecular landscape. Such information is fundamental to correlate to the potential nanotoxicology of the C3N3 nanomaterial. In this work, we explored the potential structural influence of a C3N3 nanosheet on the prototypical globular protein, villin headpiece (HP35) using all-atom molecular dynamics (MD) simulations. We found that HP35 could maintain its native conformations upon adsorption onto the C3N3 nanosheet regardless of the diversity in the binding sites, implying the potential advantage of C3N3 in protecting the biomolecular structure. The adsorption was mediated primarily by vdW interactions. Moreover, once adsorbed on the C3N3 surface, HP35 remains relatively fixed on the nanostructure without a distinct lateral translation, which may aid in keeping the structural integrity of the protein. In addition, the porous topological structure of C3N3 and the special water layer present on the C3N3 holes conjointly contributed to the restricted motion of HP35 via the formation of a high free energy barrier and a steric hindrance to prevent the surface displacement. This work revealed for the first time the potential influence of the 2D C3N3 nanomaterial in the protein structure and provided the corresponding in-depth molecular-level mechanism, which is valuable for future applications of C3N3 in bionanomedicine.

Characterization of the Clostridium perfringens phage endolysin cpp-lys and its application on lettuce.

Clostridium perfringens is an important foodborne pathogen that can have severe consequences, including mortality and economic losses. In this study, the gene encoding cpp-lys, an endolysin from the C. perfringens phage cpp has been cloned and overexpressed. The encoded protein was characterized, and then its efficacy in controlling C. perfringens on lettuce was evaluated. The endolysin cpp-lys presented lytic activity against seven strains of C. perfringens that produce different types of toxins. It maintained stability across a wide range of temperatures (4 °C - 50 °C), and demonstrated tolerance to varying pH levels (4-9). Storage of endolysin cpp-lys under room-temperature conditions (16 °C-25 °C) and cold-temperature conditions (4 °C, -20 °C, and -80 °C) for 30 days did not affect its lytic activity. However, the lytic activity of cpp-lys decreased by 40 % and 18 % after storage for 30 d at 42 °C and 37 °C, respectively. The endolysin cpp-lys did not display cytotoxic activity against normal eukaryotic cells. The bacterial viability on lettuce was significantly lower in the group treated with endolysin cpp-lys than in the PBS group, and >4-log of C. perfringens J1 were removed within 15 min. Cpp-lys plus Zn2+ inhibited the activity of cpp-lys. The EDTA-treated cpp-lys significantly reduced the number of bacteria by up to 0.6-log CFU compared with the endolysin cpp-lys group. The findings of this study demonstrated that endolysin cpp-lys has potential applications in controlling C. perfringens in the food industry.

Miniaturized kapok fiber-supported liquid extraction for convenient extraction of pesticide residues in vegetable oils: Determination of organochlorine pesticides as a proof-of-concept study.

In this paper, a miniaturized kapok fiber-supported liquid extraction (mini-KF-SLE) method was proposed for selective extraction of pesticide residues in vegetable oils. The natural kapok fiber was used as an inert oil support material based on its hydrophobic and lipophilic properties, and the extraction device was conveniently constructed by loading 15 mg of kapok fiber at the lower middle part of a 1-mL pipette tip. The vegetable oil sample (150 mg) without any pretreatment was directly loaded, followed by the addition of 150 µL of acetonitrile (ACN) as the extractant. After static extraction for 30 min, the extractant was pipetted out with a pipettor. As the proof of concept, it was applied for extracting eight organochlorine pesticides (OCPs) from vegetable oils and the eluate was analyzed by gas chromatography-electron capture detector (GC-ECD). Under optimized conditions, the extraction recoveries of OCPs were calculated to be in ranges of 35.8-79.5%. The satisfied quantitation ability was verified by the established method with coefficients of determination (R2) being greater than 0.99. The limits of detection (LODs) were in ranges of 2.0-50.0 ng/g. The relative recoveries were in ranges of 78.3-117.0% with the inter-/intra-day relative standard deviation (RSD) both being less than 13.3%. The potential of mini-KF-SLE to extract other kinds of pesticides was further verified by the successful extracting three triazole pesticides in vegetable oils with good extraction recoveries (>41.4%). The proposed mini-KF-SLE in combination with instrument detection techniques has the great potential in the low-cost and high-throughput determination of various pesticide residues in vegetable oils.

Non-Targeted Serum Lipidomics Analysis and Potential Biomarkers of Laryngeal Cancer Based on UHPLC-QTOF-MS.

Laryngeal cancer is a common head and neck malignant cancer type. However, effective biomarkers for diagnosis are lacking and pathogenesis is unclear. Lipidomics is a powerful tool for identifying biomarkers and explaining disease mechanisms. Hence, in this study, non-targeted lipidomics based on ultra-performance liquid chromatography-quadrupole time of flight-mass spectrometry (UHPLC-QTOF-MS) were applied to screen the differential lipid metabolites in serum and allowed for exploration of the remodeled lipid metabolism of laryngeal cancer, laryngeal benign tumor patients, and healthy crowds. Multivariate analysis and univariate analysis were combined to screen for differential lipid metabolites among the three groups. The results showed that, across a total of 57 lipid metabolic markers that were screened, the regulation of the lipid metabolism network occurred mainly in phosphatidylcholine (PC), lysophosphatidylcholine (LPC), and sphingomyelin (SM) metabolism. Of note, the concentration levels of sphingolipids 42:2 (SM 42:2) and sphingolipids 42:3 (SM 42:3) correlated with laryngeal cancer progression and were both significantly different among the three groups. Both of them could be considered as potential biomarkers for diagnosis and indicators for monitoring the progression of laryngeal cancer. From the perspective of lipidomics, this study not only revealed the regulatory changes in the lipid metabolism network, but also provided a new possibility for screening biomarkers in laryngeal cancer.

Development of Two Loop-Mediated Isothermal Amplification Assays for Rapid Detection of ermB and mefA Genes in Streptococcus suis.

Streptococcus suis is an important zoonotic pathogen that poses a serious threat to the pig industry and human health. The massive use of macrolides has led to the emergence of resistance in S. suis, and S. suis is suspected to be a reservoir of antimicrobial resistance genes. The mechanism to macrolide resistance in S. suis is mainly due to ermB and mefA. In this study, loop-mediated isothermal amplification (LAMP) methods were developed to detect ermB and mefA genes in S. suis through turbidimetry detection. The sensitivity and specificity of the LAMP reactions were determined. All results of LAMP and polymerase chain reaction (PCR) assay were compared to determine whether LAMP method was accurate and reliable. The results showed that all 100 nonstreptococcus clinical isolates tested negative, indicating the high specificity of LAMP assays. The detection limit of LAMP assay was 1 fg per reaction, and 102-104-fold lower than those of conventional PCR methods. Evaluation of the performance of the LAMP assay in S. suis clinical strains revealed a good consistency between LAMP and PCR assays. In conclusion, LAMP assays are specific, sensitive, and rapid methods to detect ermB and mefA in S. suis.

Kapok fiber-supported liquid extraction for convenient oil samples preparations: A feasibility and proof-of-concept study.

In this study, a novel kapok fiber-supported liquid extraction (KF-SLE) method was developed for conveniently extracting analytes from oil samples. Natural kapok fiber without any pretreatment was directly used as an oil support medium. The extraction device was conveniently constructed by directly packing some kapok fibers into a syringe tube. Due to the fibrous property of the kapok fiber, no filter plate was needed. The cost of a KF-SLE device was as low as 0.5 CNY. The KF-SLE process was conveniently conducted using a simple three-step protocol: (1) the oil sample without any pretreatment including dilution was added directedly; (2) then, the oil-immiscible extractant was added; (3) after waiting a certain time for static extraction, the extractant was eluted out by pressing the kapok fibers with the syringe plunger. The extractant could be directly transferred for subsequent instrumental detection. For the feasibility and proof-of-concept study, the method was applied to quantify four synthetic flavor chemicals in edible oils. Satisfied quantification results were obtained with the correlation coefficient (R2) being greater than 0.996, the relative recoveries ranging from 92.90% to 107.53% and intra- and inter-day RSDs being less than 7.56%. All in all, for the first time, the SLE technique was expanded to process oil samples and the method has the characteristics of low cost, environmental friendliness, high sample processing throughput and ease of automation, offering a promising approach for edible oil sample preparations.

Investigation of exposure biomarkers in human plasma following differing levels of tobacco-specific N-nitrosamines and nicotine in cigarette smoke.

Tobacco-specific N-nitrosamines (TSNAs) are strong carcinogens widely found in tobacco products, environmental tobacco smoke, lake, and wastewater. The main objective of this study was to investigate the effects of cigarette smoke with different yields of TSNAs (NNK, NNN, NAT, NAB) and nicotine on the levels of biomarkers of exposure in smokers' plasma. Three hundred healthy volunteers were recruited comprising 60 smokers of each of 3 mg, 8 mg and 10 mg ISO tar yield cigarettes and 60 smokers who smoked 10 mg, 8 mg, and 3 mg for 14 days sequentially and 60 non-smokers. All study participants were male, aged from 21 to 45 years old, and were recruited from a same unit in Hebei, China. We measured the levels of NNAL, NAT, NNN, NAB and cotinine in plasma from 240 smokers and 60 non-smokers using a novel method established by online two-dimensional solid phase extraction-liquid chromatography-tandem mass spectrometry. The results showed that NNAL, NAT, NNN, NAB and cotinine in the plasma of smokers smoking cigarette with low TSNAs and nicotine were lower than that with high TSNAs and nicotine. When smokers switched from higher to lower TSNA yields of cigarettes, their plasma NNAL, NAT, NNN, NAB levels significantly decreased. The plasma concentrations of NNAL were significantly correlated with those of cotinine, NNN, NAT and NAB for smokers (p < 0.001). Similarly, the plasma concentrations of cotinine were significantly correlated with those of NNN, NAT and NAB for smokers (p < 0.001). The plasma NNAL, NAT, NNN, NAB and cotinine levels for smokers were significantly higher than those for non-smokers. These findings suggested that the total NNAL, NNN, NAT, NAB and cotinine in plasma were valid and reliable biomarkers for human exposure to TSNAs and nicotine.

Diagnosis and treatment of diverticular hemorrhage in small intestine: A retrospective study.

BACKGROUND AND STUDY AIMS: Small intestine diverticula are the most common cause of gastrointestinal hemorrhage, but prompt diagnosis remains challenging. Thus, this study aimed to identify strategies for the diagnosis and treatment of diverticular hemorrhage. PATIENTS AND METHODS: Patients who presented with gastrointestinal tract bleeding to Guangzhou First People's Hospital between 2008 and 2014 were retrospectively examined. Gastrorrhagia and colonic hemorrhage were excluded based on the gastroscopy and colonoscopy findings, and the bleeding sites were in the small intestine. Data regarding patient characteristics, methods of diagnosis, treatment, and prognosis were collected. RESULTS: Eighty-five patients met the study criteria, and 45 patients were diagnosed with diverticular hemorrhage using double balloon enteroscopy, capsule endoscopy, computed tomography (CT), or digital subtraction angiography (DSA). Among these patients, 10 presented with massive bleeding and hemodynamic instability. All 45 patients underwent surgery and recovered with no complications, and all patients were followed-up for over 3 years, with no cases of recurrent hemorrhage. CONCLUSION: Diverticular hemorrhage is the most common cause of small intestine bleeding. Double balloon enteroscopy, capsule endoscopy, CT, and DSA are effective methods for diagnosing small intestine diverticular hemorrhage. Surgical resection of the involved region is necessary and may achieve complete cure.

High throughput and very specific screening of anabolic-androgenic steroid adulterants in healthy foods based on stable isotope labelling and flow injection analysis-tandem mass spectrometry with simultaneous monitoring proton adduct ions and chloride adduct ions.

In this work, a stable isotope labelling-flow injection analysis-tandem mass spectrometry (SIL-FIA-MS/MS) with simultaneous monitoring [M+H]+ and [M+Cl]- method was developed for very specific and high throughput screening of anabolic-androgenic steroids (AAS) illegally added to healthy foods. Initially, a simple centrifugation step was carried out for liquid samples, and for solid samples, a solid-liquid extraction step was conducted. Afterwards, batch chemical derivatization was carried out. After adding a certain amount of 13C6-3-NPH labelled AAS standards as the internal standards, it can be directly transferred for FIA-MS/MS analysis based on the no MS response characteristics of 3-NPH. The 3-NPH labelled AAS showed dual-polarity property, observing chloride adduct ion ([M+Cl]-) in negative ion mode and proton adduct ion ([M+H]+) in positive ion mode. The average time cost for pretreatment of each sample was less than 1 min by carrying out batch processing. The subsequent FIA-MS/MS detection enabled rapid and high throughput detection. The addition of 13C6-3-NPH-labelled AAS as internal standards can correct the matrix effect to achieve accurate quantitative analysis. The detection sensitivity was also improved by 2-5 folds after 3-NPH labelling. The limits of detection (LODs) in positive MRM mode were in ranges of 0.1-0.3 ng/mL. The validated method with simultaneous monitoring [M+H]+ and [M+Cl]- was validated in the range of 6.0-1000 ng/mL with the linear coefficient (R2) greater than 0.997. Satisfactory recoveries were found to be in ranges of 93.0-108.7%. The intra-day and inter-day RSDs were in the range of 3.5-9.9% and 5.1-14.1%, respectively. No changes in detection sensitivity of the mass spectrometry and no carry-over effects were found after numerous consecutive injections of AAS derivates. Compared with previously reported methods, the proposed method proved accurate, very specific, high throughput with good sensitivity.

Whole-Genome Epidemiology and Characterization of Methicillin-Susceptible Staphylococcus aureus ST398 From Retail Pork and Bulk Tank Milk in Shandong, China.

Staphylococcus aureus (S. aureus) is now regarded as a zoonotic agent. Methicillin-susceptible S. aureus (MSSA) ST398 is a livestock-associated bacterium that is most prevalent in China, but there are currently no data available for Shandong. Therefore, the aim of this study was to investigate the epidemiology and characterization of MSSA ST398 from retail pork and bulk tank milk (BTM) in Shandong. A total of 67 S. aureus isolates were collected from retail pork between November 2017 and June 2018. Among the isolates, high antimicrobial resistance rates were observed for penicillin (97.0%), and 92.5% of the isolates were multi-drug resistant (MDR). Eight sequence types (STs) were identified in the retail pork isolates, and the predominant type was ST15 (n=26), which was followed by ST398 (n=14). Staphylococcal protein A gene (spa) typing identified spa types t034 and t1255 in MSSA ST398 from retail pork. Using whole-genome sequencing analysis, we described the phylogeny of 29 MSSA ST398 isolates that were obtained from retail pork (n=14) and BTM (n=15). The phylogenetic tree showed that the MSSA ST398 isolates from different sources had the same lineage. Among the 29 MSSA ST398 isolates, five resistance genes were detected, and all isolates carried DHA-1. Fifteen toxin genes were detected, and all isolates carried eta, hla, and hlb. In conclusion, this study found that a high risk for MSSA ST398 was present in retail pork and BTM. These findings have major implications for how investigations of MSSA ST398 outbreaks should be conducted in the One-Health context.